Human skeletal muscle methylome after low-carbohydrate energy-balanced exercise.

We aimed to investigate the human skeletal muscle (SkM) DNA methylome after exercise in low-carbohydrate (CHO) energy-balance (with high-fat) conditions compared with exercise in low-CHO energy-deficit (with low-fat) conditions. The objective was to identify novel epigenetically regulated genes and pathways associated with "train-low sleep-low" paradigms. The sleep-low conditions included nine males that cycled to deplete muscle glycogen while reaching a set energy expenditure. Postexercise, low-CHO meals (protein matched) completely replaced (using high fat) or only partially replaced (low fat) the energy expended. The following morning, resting baseline biopsies were taken and the participants then undertook 75 minutes of cycling exercise, with skeletal muscle biopsies collected 30 minutes and 3.5 hours postexercise. Discovery of genome-wide DNA methylation was undertaken using Illumina EPIC arrays, and targeted gene expression analysis was conducted by quantitative RT-PCR. At baseline, participants under energy balance (high fat) demonstrated a predominantly hypermethylated (60%) profile across the genome compared to energy-deficit low-fat conditions. However, postexercise performed in energy balance (with high fat) elicited a more prominent hypomethylation signature 30 minutes postexercise in gene regulatory regions important for transcription (CpG islands within promoter regions) compared with exercise in energy-deficit (with low-fat) conditions. Such hypomethylation was enriched within pathways related to IL6-JAK-STAT signaling, metabolic processes, p53/cell cycle, and oxidative/fatty acid metabolism. Hypomethylation within the promoter regions of the genes; histone deacetylase 2 (HDAC2), MECR, IGF2, and c13orf16 were associated with significant increases in gene expression in the postexercise period in energy balance compared with an energy deficit. Furthermore, HDAC11 was oppositely regulated at the gene expression level compared with family member HDAC2, where HDAC11 was hypomethylated yet increased in energy-deficit compared with energy-balance conditions. Overall, we identify some novel epigenetically regulated genes associated with train-low sleep-low paradigms.NEW & NOTEWORTHY We identify novel epigenetically regulated genes associated with train-low sleep-low paradigms. Exercise under low-carbohydrate (CHO) energy-balance (high-fat) conditions elicited a more prominent DNA hypomethylation signature 30 minutes postexercise compared with low-CHO energy-deficit (low-fat) conditions. This was enriched within IL6-JAK-STAT signaling, metabolic processes, p53, cell cycle, oxidative phosphorylation, and fatty acid metabolism. Histone deacetylase (HDAC) family members 2, 4, 10, and 11 demonstrated hypomethylation, with HDAC2 and HDAC11 possessing alternative regulation of gene expression in energy balance versus deficit conditions.

Aerobic exercise training resets the human skeletal muscle methylome 10 years after breast cancer treatment and survival.

Cancer survivors suffer impairments in skeletal muscle in terms of reduced mass and function. Interestingly, human skeletal muscle possesses an epigenetic memory of earlier stimuli, such as exercise. Long-term retention of epigenetic changes in skeletal muscle following cancer survival and/or exercise training has not yet been studied. We, therefore, investigated genome-wide DNA methylation (methylome) in skeletal muscle following a 5-month, 3/week aerobic-training intervention in breast cancer survivors 10-14 years after diagnosis and treatment. These results were compared to breast cancer survivors who remained untrained and to age-matched controls with no history of cancer, who undertook the same training intervention. Skeletal muscle biopsies were obtained from 23 females before(pre) and after(post) the 5-month training period. InfiniumEPIC 850K DNA methylation arrays and RT-PCR for gene expression were performed. The breast cancer survivors displayed a significant retention of increased DNA methylation (i.e., hypermethylation) at a larger number of differentially methylated positions (DMPs) compared with healthy age-matched controls pre training. Training in cancer survivors led to an exaggerated number of DMPs with a hypermethylated signature occurring at non-regulatory regions compared with training in healthy age-matched controls. However, the opposite occurred in important gene regulatory regions, where training in cancer survivors elicited a considerable reduction in methylation (i.e., hypomethylation) in 99% of the DMPs located in CpG islands within promoter regions. Importantly, training was able to reverse the hypermethylation identified in cancer survivors back toward a hypomethylated signature that was observed pre training in healthy age-matched controls at 300 (out of 881) of these island/promoter-associated CpGs. Pathway enrichment analysis identified training in cancer survivors evoked a predominantly hypomethylated signature in pathways associated with cell cycle, DNA replication/repair, transcription, translation, mTOR signaling, and the proteosome. Differentially methylated region (DMR) analysis also identified genes: BAG1, BTG2, CHP1, KIFC1, MKL2, MTR, PEX11B, POLD2, S100A6, SNORD104, and SPG7 as hypermethylated in breast cancer survivors, with training reversing these CpG island/promoter-associated DMRs toward a hypomethylated signature. Training also elicited a largely different epigenetic response in healthy individuals than that observed in cancer survivors, with very few overlapping changes. Only one gene, SIRT2, was identified as having altered methylation in cancer survivors at baseline and after training in both the cancer survivors and healthy controls. Overall, human skeletal muscle may retain a hypermethylated signature as long as 10-14 years after breast cancer treatment/survival. Five months of aerobic training reset the skeletal muscle methylome toward signatures identified in healthy age-matched individuals in gene regulatory regions.

Aging, Skeletal Muscle, and Epigenetics.

SUMMARY: We are living in an aging society. In 2019, 1 billion individuals were already aged over 60. The number of people in this demographic is predicted to reach 1.4 billion by 2030 and 2.1 billion by 2050 (WHO). In the USA, individuals over 65 represent the fastest growing segment of the population (US census bureau). Similar trends are seen in the UK, with 16.2 million people already aged over 60, equivalent to 24% of the total population (Age UK; https://www.ageuk.org.uk/globalassets/age-uk/documents/reports-and-publications/later_life_uk_factsheet.pdf). Indeed, in the UK, people over the age of 60 outnumbered those under the age of 18, for the first time in 2008. This statistic still prevails today. Because of medical and biopharmaceutical progress, lifespan is increasing rapidly, but healthspan is failing to keep up. If we are to increase healthy living, then we need to begin to understand the mechanisms of how we age across the life course, so that relevant interventions may be developed to facilitate "life in our years," not simply "years in our life." It is reported that only 25% of aging is genetically predetermined. This fits with observations of some families aging very quickly and poorly and others aging slowly and well. If this is indeed the case and the rate of aging is not fixed, then this knowledge provides a significant opportunity to manipulate the impact of environmental influencers of age. With that in mind, it begs the question of what are the mechanisms of aging and is there potential to manipulate this process on an individual-by-individual basis? The focus of this article will be on the process of muscle wasting with aging (sarcopenia) and the potential of exercise and its underlying mechanisms to reverse or delay sarcopenia. There will be a focus on epigenetics in muscle wasting and the capability of exercise to change our skeletal muscle epigenetic profile for the good. The article ends with considerations relating to facial aging, Botox treatment, and gene editing as a tool for plastic surgeons in the future.

Meta-analysis of genome-wide DNA methylation and integrative omics of age in human skeletal muscle.

BACKGROUND: Knowledge of age-related DNA methylation changes in skeletal muscle is limited, yet this tissue is severely affected by ageing in humans. METHODS: We conducted a large-scale epigenome-wide association study meta-analysis of age in human skeletal muscle from 10 studies (total n = 908 muscle methylomes from men and women aged 18-89 years old). We explored the genomic context of age-related DNA methylation changes in chromatin states, CpG islands, and transcription factor binding sites and performed gene set enrichment analysis. We then integrated the DNA methylation data with known transcriptomic and proteomic age-related changes in skeletal muscle. Finally, we updated our recently developed muscle epigenetic clock (https://bioconductor.org/packages/release/bioc/html/MEAT.html). RESULTS: We identified 6710 differentially methylated regions at a stringent false discovery rate <0.005, spanning 6367 unique genes, many of which related to skeletal muscle structure and development. We found a strong increase in DNA methylation at Polycomb target genes and bivalent chromatin domains and a concomitant decrease in DNA methylation at enhancers. Most differentially methylated genes were not altered at the mRNA or protein level, but they were nonetheless strongly enriched for genes showing age-related differential mRNA and protein expression. After adding a substantial number of samples from five datasets (+371), the updated version of the muscle clock (MEAT 2.0, total n = 1053 samples) performed similarly to the original version of the muscle clock (median of 4.4 vs. 4.6 years in age prediction error), suggesting that the original version of the muscle clock was very accurate. CONCLUSIONS: We provide here the most comprehensive picture of DNA methylation ageing in human skeletal muscle and reveal widespread alterations of genes involved in skeletal muscle structure, development, and differentiation. We have made our results available as an open-access, user-friendly, web-based tool called MetaMeth (https://sarah-voisin.shinyapps.io/MetaMeth/).

Knockdown of the E3 ubiquitin ligase UBR5 and its role in skeletal muscle anabolism.

UBR5 is an E3 ubiquitin ligase positively associated with anabolism, hypertrophy, and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5's role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in vivo. To achieve this aim, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle to investigate the impact of reduced UBR5 on anabolic signaling MEK/ERK/p90RSK and Akt/GSK3ß/p70S6K/4E-BP1/rpS6 pathways. Seven days after UBR5 RNAi electroporation, although reductions in overall muscle mass were not detected, the mean cross-sectional area (CSA) of green fluorescent protein (GFP)-positive fibers were reduced (-9.5%) and the number of large fibers were lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2, Akt, and GSK3ß activity. Although p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (-4.6%) and larger reductions in fiber CSA (-18.5%) after 30 days. This was associated with increased levels of phosphatase PP2Ac and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, as well as corresponding reductions in eIF4e. This study demonstrates that UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

An epigenetic clock for human skeletal muscle.

BACKGROUND: Ageing is associated with DNA methylation changes in all human tissues, and epigenetic markers can estimate chronological age based on DNA methylation patterns across tissues. However, the construction of the original pan-tissue epigenetic clock did not include skeletal muscle samples and hence exhibited a strong deviation between DNA methylation and chronological age in this tissue. METHODS: To address this, we developed a more accurate, muscle-specific epigenetic clock based on the genome-wide DNA methylation data of 682 skeletal muscle samples from 12 independent datasets (18-89 years old, 22% women, 99% Caucasian), all generated with Illumina HumanMethylation (HM) arrays (HM27, HM450, or HMEPIC). We also took advantage of the large number of samples to conduct an epigenome-wide association study of age-associated DNA methylation patterns in skeletal muscle. RESULTS: The newly developed clock uses 200 cytosine-phosphate-guanine dinucleotides to estimate chronological age in skeletal muscle, 16 of which are in common with the 353 cytosine-phosphate-guanine dinucleotides of the pan-tissue clock. The muscle clock outperformed the pan-tissue clock, with a median error of only 4.6 years across datasets (vs. 13.1 years for the pan-tissue clock, P < 0.0001) and an average correlation of ρ = 0.62 between actual and predicted age across datasets (vs. ρ = 0.51 for the pan-tissue clock). Lastly, we identified 180 differentially methylated regions with age in skeletal muscle at a false discovery rate < 0.005. However, gene set enrichment analysis did not reveal any enrichment for gene ontologies. CONCLUSIONS: We have developed a muscle-specific epigenetic clock that predicts age with better accuracy than the pan-tissue clock. We implemented the muscle clock in an r package called Muscle Epigenetic Age Test available on Bioconductor to estimate epigenetic age in skeletal muscle samples. This clock may prove valuable in assessing the impact of environmental factors, such as exercise and diet, on muscle-specific biological ageing processes.

Comparative Transcriptome and Methylome Analysis in Human Skeletal Muscle Anabolism, Hypertrophy and Epigenetic Memory.

Transcriptome wide changes in human skeletal muscle after acute (anabolic) and chronic resistance exercise (RE) induced hypertrophy have been extensively determined in the literature. We have also recently undertaken DNA methylome analysis (850,000 + CpG sites) in human skeletal muscle after acute and chronic RE, detraining and retraining, where we identified an association between DNA methylation and epigenetic memory of exercise induced skeletal muscle hypertrophy. However, it is currently unknown as to whether all the genes identified in the transcriptome studies to date are also epigenetically regulated at the DNA level after acute, chronic or repeated RE exposure. We therefore aimed to undertake large scale bioinformatical analysis by pooling the publicly available transcriptome data after acute (110 samples) and chronic RE (181 samples) and comparing these large data sets with our genome-wide DNA methylation analysis in human skeletal muscle after acute and chronic RE, detraining and retraining. Indeed, after acute RE we identified 866 up- and 936 down-regulated genes at the expression level, with 270 (out of the 866 up-regulated) identified as being hypomethylated, and 216 (out of 936 downregulated) as hypermethylated. After chronic RE we identified 2,018 up- and 430 down-regulated genes with 592 (out of 2,018 upregulated) identified as being hypomethylated and 98 (out of 430 genes downregulated) as hypermethylated. After KEGG pathway analysis, genes associated with 'cancer' pathways were significantly enriched in both bioinformatic analysis of the pooled transcriptome and methylome datasets after both acute and chronic RE. This resulted in 23 (out of 69) and 28 (out of 49) upregulated and hypomethylated and 12 (out of 37) and 2 (out of 4) downregulated and hypermethylated 'cancer' genes following acute and chronic RE respectively. Within skeletal muscle tissue, these 'cancer' genes predominant functions were associated with matrix/actin structure and remodelling, mechano-transduction (e.g. PTK2/Focal Adhesion Kinase and Phospholipase D- following chronic RE), TGF-beta signalling and protein synthesis (e.g. GSK3B after acute RE). Interestingly, 51 genes were also identified to be up/downregulated in both the acute and chronic RE pooled transcriptome analysis as well as significantly hypo/hypermethylated after acute RE, chronic RE, detraining and retraining. Five genes; FLNB, MYH9, SRGAP1, SRGN, ZMIZ1 demonstrated increased gene expression in the acute and chronic RE transcriptome and also demonstrated hypomethylation in these conditions. Importantly, these 5 genes demonstrated retained hypomethylation even during detraining (following training induced hypertrophy) when exercise was ceased and lean mass returned to baseline (pre-training) levels, identifying them as genes associated with epigenetic memory in skeletal muscle. Importantly, for the first time across the transcriptome and epigenome combined, this study identifies novel differentially methylated genes associated with human skeletal muscle anabolism, hypertrophy and epigenetic memory.

Exercising Bioengineered Skeletal Muscle In Vitro: Biopsy to Bioreactor.

The bioengineering of skeletal muscle tissue in-vitro has enabled researchers to more closely mimic the in-vivo skeletal muscle niche. The three-dimensional (3-D) structure of the tissue engineered systems employed to date enable the generation of highly aligned and differentiated myofibers within a representative biological matrix. The use of electrical stimulation to model concentric contraction, via innervation of the myofibers, and the use of mechanical loading to model passive lengthening or stretch has begun to provide a manipulable environment to investigate the cellular and molecular responses following exercise mimicking stimuli in-vitro. Currently available bioreactor systems allow either electrical stimulation or mechanical loading to be utilized at any given time. In the present manuscript, we describe in detail the methodological procedures to create 3-D bioengineered skeletal muscle using both cell lines and/or primary human muscle derived cells from a tissue biopsy, through to modeling exercising stimuli using a bioreactor that can provide both electrical stimulation and mechanical loading simultaneously within the same in-vitro system.

Whey Protein Augments Leucinemia and Postexercise p70S6K1 Activity Compared With a Hydrolyzed Collagen Blend When in Recovery From Training With Low Carbohydrate Availability.

We examined the effects of whey versus collagen protein on skeletal muscle cell signaling responses associated with mitochondrial biogenesis and protein synthesis in recovery from an acute training session completed with low carbohydrate availability. In a repeated-measures design (after adhering to a 36-hr exercise-dietary intervention to standardize preexercise muscle glycogen), eight males completed a 75-min nonexhaustive cycling protocol and consumed 22 g of a hydrolyzed collagen blend (COLLAGEN) or whey (WHEY) protein 45 min prior to exercise, 22 g during exercise, and 22 g immediately postexercise. Exercise decreased (p < .05) muscle glycogen content by comparable levels from pre- to postexercise in both trials (≈300-150 mmol/kg·dry weight). WHEY protein induced greater increases in plasma branched chain amino acids (p = .03) and leucine (p = .02) than COLLAGEN. Exercise induced (p < .05) similar increases in PGC-1α (fivefold) mRNA at 1.5 hr postexercise between conditions, although no effect of exercise (p > .05) was observed for p53, Parkin, and Beclin1 mRNA. Exercise suppressed (p < .05) p70S6K1 activity in both conditions immediately postexercise (≈25 fmol·min-1·mg-1). Postexercise feeding increased p70S6K1 activity at 1.5 hr postexercise (p < .05), the magnitude of which was greater (p < .05) in WHEY (180 ± 105 fmol·min-1·mg-1) versus COLLAGEN (73 ± 42 fmol·min-1·mg-1). We conclude that protein composition does not modulate markers of mitochondrial biogenesis when in recovery from a training session deliberately completed with low carbohydrate availability. By contrast, whey protein augments postexercise p70S6K activity compared with hydrolyzed collagen, as likely mediated via increased leucine availability.

Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

Physical inactivity and disuse are major contributors to age-related muscle loss. Denervation of skeletal muscle has been previously used as a model with which to investigate muscle atrophy following disuse. Although gene regulatory networks that control skeletal muscle atrophy after denervation have been established, the transcriptome in response to the recovery of muscle after disuse and the associated epigenetic mechanisms that may function to modulate gene expression during skeletal muscle atrophy or recovery have yet to be investigated. We report that silencing the tibialis anterior muscle in rats with tetrodotoxin (TTX)-administered to the common peroneal nerve-resulted in reductions in muscle mass of 7, 29, and 51% with corresponding reductions in muscle fiber cross-sectional area of 18, 42, and 69% after 3, 7, and 14 d of TTX, respectively. Of importance, 7 d of recovery, during which rodents resumed habitual physical activity, restored muscle mass from a reduction of 51% after 14 d TTX to a reduction of only 24% compared with sham control. Returning muscle mass to levels observed at 7 d TTX administration (29% reduction). Transcriptome-wide analysis demonstrated that 3714 genes were differentially expressed across all conditions at a significance of P ≤ 0.001 after disuse-induced atrophy. Of interest, after 7 d of recovery, the expression of genes that were most changed during TTX had returned to that of the sham control. The 20 most differentially expressed genes after microarray analysis were identified across all conditions and were cross-referenced with the most frequently occurring differentially expressed genes between conditions. This gene subset included myogenin (MyoG), Hdac4, Ampd3, Trim63 (MuRF1), and acetylcholine receptor subunit α1 (Chrna1). Transcript expression of these genes and Fboxo32 (MAFbx), because of its previously identified role in disuse atrophy together with Trim63 (MuRF1), were confirmed by real-time quantitative RT-PCR, and DNA methylation of their promoter regions was analyzed by PCR and pyrosequencing. MyoG, Trim63 (MuRF1), Fbxo32 (MAFbx), and Chrna1 demonstrated significantly decreased DNA methylation at key time points after disuse-induced atrophy that corresponded with significantly increased gene expression. Of importance, after TTX cessation and 7 d of recovery, there was a marked increase in the DNA methylation profiles of Trim63 (MuRF1) and Chrna1 back to control levels. This also corresponded with the return of gene expression in the recovery group back to baseline expression observed in sham-surgery controls. To our knowledge, this is the first study to demonstrate that skeletal muscle atrophy in response to disuse is accompanied by dynamic epigenetic modifications that are associated with alterations in gene expression, and that these epigenetic modifications and gene expression profiles are reversible after skeletal muscle returns to normal activity.-Fisher, A. G., Seaborne, R. A., Hughes, T. M., Gutteridge, A., Stewart, C., Coulson, J. M., Sharples, A. P., Jarvis, J. C. Transcriptomic and epigenetic regulation of disuse atrophy and the return to activity in skeletal muscle.

Omega-3 fatty acid EPA improves regenerative capacity of mouse skeletal muscle cells exposed to saturated fat and inflammation.

Sarcopenic obesity is characterised by high fat mass, low muscle mass and an elevated inflammatory environmental milieu. We therefore investigated the effects of elevated inflammatory cytokine TNF-α (aging/obesity) and saturated fatty acid, palmitate (obesity) on skeletal muscle cells in the presence/absence of EPA, a-3 polyunsaturated fatty acid with proposed anti-inflammatory, anti-obesity activities. In the present study we show that palmitate was lipotoxic, inducing high levels of cell death and blocking myotube formation. Cell death under these conditions was associated with increased caspase activity, suppression of differentiation, reductions in both creatine kinase activity and gene expression of myogenic factors; IGF-II, IGFBP-5, MyoD and myogenin. However, inhibition of caspase activity via administration of Z-VDVAD-FMK (caspase-2), Z-DEVD-FMK (caspase-3) and ZIETD-KMK (caspase 8) was without effect on cell death. By contrast, lipotoxicity associated with elevated palmitate was reduced with the MEK inhibitor PD98059, indicating palmitate induced cell death was MAPK mediated. These lipotoxic conditions were further exacerbated in the presence of inflammation via TNF-α co-administration. Addition of EPA under cytotoxic stress (TNF-α) was shown to partially rescue differentiation with enhanced myotube formation being associated with increased MyoD, myogenin, IGF-II and IGFBP-5 expression. EPA had little impact on the cell death phenotype observed in lipotoxic conditions but did show benefit in restoring differentiation under lipotoxic plus cytotoxic conditions. Under these conditions Id3 (inhibitor of differentiation) gene expression was inversely linked with survival rates, potentially indicating a novel role of EPA and Id3 in the regulation of apoptosis in lipotoxic/cytotoxic conditions. Additionally, signalling studies indicated the combination of lipo- and cyto-toxic effects on the muscle cells acted through ceramide, JNK and MAPK pathways and blocking these pathways using PD98059 (MEK inhibitor) and Fumonisin B1 (ceramide inhibitor) significantly reduced levels of cell death. These findings highlight novel pathways associated with in vitro models of lipotoxicity (palmitate-mediated) and cytotoxicity (inflammatory cytokine mediated) in the potential targeting of molecular modulators of sarcopenic obesity.

Postexercise High-Fat Feeding Suppresses p70S6K1 Activity in Human Skeletal Muscle.

PURPOSE: This study aimed to examine the effects of reduced CHO but high postexercise fat availability on cell signaling and expression of genes with putative roles in regulation of mitochondrial biogenesis, lipid metabolism, and muscle protein synthesis. METHODS: Ten males completed a twice per day exercise model (3.5 h between sessions) comprising morning high-intensity interval training (8 × 5 min at 85% V˙O2peak) and afternoon steady-state (SS) running (60 min at 70% V˙O2peak). In a repeated-measures design, runners exercised under different isoenergetic dietary conditions consisting of high-CHO (HCHO: 10 g·kg CHO, 2.5 g·kg protein, and 0.8 g·kg fat for the entire trial period) or reduced-CHO but high-fat availability in the postexercise recovery periods (HFAT: 2.5 g·kg CHO, 2.5 g·kg protein, and 3.5 g·kg fat for the entire trial period). RESULTS: Muscle glycogen was lower (P < 0.05) at 3 h (251 vs 301 mmol·kg dry weight) and 15 h (182 vs 312 mmol·kg dry weight) post-SS exercise in HFAT compared with HCHO. Adenosine monophosphate-activated protein kinase α2 activity was not increased post-SS in either condition (P = 0.41), although comparable increases (all P < 0.05) in PGC-1α, p53, citrate synthase, Tfam, peroxisome proliferator-activated receptor, and estrogen-related receptor α mRNA were observed in HCHO and HFAT. By contrast, PDK4 (P = 0.003), CD36 (P = 0.05), and carnitine palmitoyltransferase 1 (P = 0.03) mRNA were greater in HFAT in the recovery period from SS exercise compared with HCHO. Ribosomal protein S6 kinase activity was higher (P = 0.08) at 3 h post-SS exercise in HCHO versus HFAT (72.7 ± 51.9 vs 44.7 ± 27 fmol·min·mg). CONCLUSION: Postexercise high-fat feeding does not augment the mRNA expression of genes associated with regulatory roles in mitochondrial biogenesis, although it does increase lipid gene expression. However, postexercise ribosomal protein S6 kinase 1 activity is reduced under conditions of high-fat feeding, thus potentially impairing skeletal muscle remodeling processes.

Does skeletal muscle have an 'epi'-memory? The role of epigenetics in nutritional programming, metabolic disease, aging and exercise.

Skeletal muscle mass, quality and adaptability are fundamental in promoting muscle performance, maintaining metabolic function and supporting longevity and healthspan. Skeletal muscle is programmable and can 'remember' early-life metabolic stimuli affecting its function in adult life. In this review, the authors pose the question as to whether skeletal muscle has an 'epi'-memory? Following an initial encounter with an environmental stimulus, we discuss the underlying molecular and epigenetic mechanisms enabling skeletal muscle to adapt, should it re-encounter the stimulus in later life. We also define skeletal muscle memory and outline the scientific literature contributing to this field. Furthermore, we review the evidence for early-life nutrient stress and low birth weight in animals and human cohort studies, respectively, and discuss the underlying molecular mechanisms culminating in skeletal muscle dysfunction, metabolic disease and loss of skeletal muscle mass across the lifespan. We also summarize and discuss studies that isolate muscle stem cells from different environmental niches in vivo (physically active, diabetic, cachectic, aged) and how they reportedly remember this environment once isolated in vitro. Finally, we will outline the molecular and epigenetic mechanisms underlying skeletal muscle memory and review the epigenetic regulation of exercise-induced skeletal muscle adaptation, highlighting exercise interventions as suitable models to investigate skeletal muscle memory in humans. We believe that understanding the 'epi'-memory of skeletal muscle will enable the next generation of targeted therapies to promote muscle growth and reduce muscle loss to enable healthy aging.

l-glutamine Improves Skeletal Muscle Cell Differentiation and Prevents Myotube Atrophy After Cytokine (TNF-α) Stress Via Reduced p38 MAPK Signal Transduction.

Tumour Necrosis Factor-Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As l-glutamine can dampen the effects of inflamed environments, we investigated the role of l-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng · ml(-1) ) ± l-glutamine (20 mM). TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM l-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, l-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720-2732, 2016. © 2016 Wiley Periodicals, Inc.

Skeletal muscle cells possess a 'memory' of acute early life TNF-α exposure: role of epigenetic adaptation.

Sufficient quantity and quality of skeletal muscle is required to maintain lifespan and healthspan into older age. The concept of skeletal muscle programming/memory has been suggested to contribute to accelerated muscle decline in the elderly in association with early life stress such as fetal malnutrition. Further, muscle cells in vitro appear to remember the in vivo environments from which they are derived (e.g. cancer, obesity, type II diabetes, physical inactivity and nutrient restriction). Tumour-necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is chronically elevated in sarcopenia and cancer cachexia. Higher TNF-α levels are strongly correlated with muscle loss, reduced strength and therefore morbidity and earlier mortality. We have extensively shown that TNF-α impairs regenerative capacity in mouse and human muscle derived stem cells [Meadows et al. (J Cell Physiol 183(3):330-337, 2000); Foulstone et al. (J Cell Physiol 189(2):207-215, 2001); Foulstone et al. (Exp Cell Res 294(1):223-235, 2004); Stewart et al. (J Cell Physiol 198(2):237-247, 2004); Al-Shanti et al. (Growth factors (Chur, Switzerland) 26(2):61-73, 2008); Saini et al. (Growth factors (Chur, Switzerland) 26(5):239-253, 2008); Sharples et al. (J Cell Physiol 225(1):240-250, 2010)]. We have also recently established an epigenetically mediated mechanism (SIRT1-histone deacetylase) regulating survival of myoblasts in the presence of TNF-α [Saini et al. (Exp Physiol 97(3):400-418, 2012)]. We therefore wished to extend this work in relation to muscle memory of catabolic stimuli and the potential underlying epigenetic modulation of muscle loss. To enable this aim; C2C12 myoblasts were cultured in the absence or presence of early TNF-α (early proliferative lifespan) followed by 30 population doublings in the absence of TNF-α, prior to the induction of differentiation in low serum media (LSM) in the absence or presence of late TNF-α (late proliferative lifespan). The cells that received an early plus late lifespan dose of TNF-α exhibited reduced morphological (myotube number) and biochemical (creatine kinase activity) differentiation vs. control cells that underwent the same number of proliferative divisions but only a later life encounter with TNF-α. This suggested that muscle cells had a morphological memory of the acute early lifespan TNF-α encounter. Importantly, methylation of myoD CpG islands were increased in the early TNF-α cells, 30 population doublings later, suggesting that even after an acute encounter with TNF-α, the cells have the capability of retaining elevated methylation for at least 30 cellular divisions. Despite these fascinating findings, there were no further increases in myoD methylation or changes in its gene expression when these cells were exposed to a later TNF-α dose suggesting that this was not directly responsible for the decline in differentiation observed. In conclusion, data suggest that elevated myoD methylation is retained throughout muscle cells proliferative lifespan as result of early life TNF-α treatment and has implications for the epigenetic control of muscle loss.

Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake.

Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio-economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin-like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS-1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.

Impaired hypertrophy in myoblasts is improved with testosterone administration.

We investigated the ability of testosterone (T) to restore differentiation in multiple population doubled (PD) murine myoblasts, previously shown to have a reduced differentiation in monolayer and bioengineered skeletal muscle cultures vs. their parental controls (CON) (Sharples et al., 2011, 2012 [7,26]). Cells were exposed to low serum conditions in the presence or absence of T (100nM)±PI3K inhibitor (LY294002) for 72h and 7 days (early and late muscle differentiation respectively). Morphological analyses were performed to determine myotube number, diameter (µm) and myonuclear accretion as indices of differentiation and myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were also performed. Myotube diameter in CON and PD cells increased from 17.32±2.56µm to 21.02±1.89µm and 14.58±2.66µm to 18.29±3.08µm (P≤0.05) respectively after 72h of T exposure. The increase was comparable in both PD (+25%) and CON cells (+21%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in both cell types after 7 days exposure (P≤0.05). Addition of PI3K inhibitor (LY294002) in the presence of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also reduced myostatin expression in PD myoblasts only. The present study demonstrates testosterone administration improves hypertrophy in myoblasts that basally display impaired differentiation and hypertrophic capacity vs. their parental controls, the action of testosterone in this model was mediated by PI3K/Akt pathway.

The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy.

The complex actions of the insulin-like-growth factor binding proteins (IGFBPs) in skeletal muscle are becoming apparent, with IGFBP2 being implicated in skeletal muscle cell proliferation and differentiation (Ernst et al., 1992; Sharples et al., 2010). Furthermore, PTEN signalling has been linked to IGFBP2 action in other cell types by co-ordinating downstream Akt signalling, a known modulator of myoblast differentiation. The present study therefore aimed to determine the interaction between IGFBP2 and PTEN on myoblast differentiation. It has previously been established that C2C12 cells have high IGFBP2 gene expression upon transfer to low serum media, and that expression reduces rapidly as cells differentiate over 72 h [1]. Wishing to establish a potential role for IGFBP2 in this model, a neutralising IGFBP2 antibody was administered to C2C12 myoblasts upon initiation of differentiation. Myoblasts subsequently displayed reduced morphological differentiation (myotube number), biochemical differentiation (creatine kinase) and myotube hypertrophy (myotube area) with an early reduction in Akt phosphorylation. Knock-down of phosphatase and tensin homologue (PTEN) using siRNA in the absence of the neutralising antibody did not improve differentiation or hypertrophy vs. control conditions, however, in the presence of the neutralising IGFBP2 antibody, differentiation was restored and importantly hypertrophy exceeded that of control levels. Overall, these data suggest that; 1) reduced early availability of IGFBP2 can inhibit myoblast differentiation at later time points, 2) knock-down of PTEN levels can restore myoblast differentiation in the presence of neutralising IGFBP2 antibody, and 3) PTEN inhibition acts as a potent inducer of myotube hypertrophy when the availability of IGFBP2 is reduced in C2C12 myoblasts.

Modelling in vivo skeletal muscle ageing in vitro using three-dimensional bioengineered constructs.

Degeneration of skeletal muscle (SkM) with age (sarcopenia) is a major contributor to functional decline, morbidity and mortality. Methodological implications often make it difficult to embark on interventions in already frail and diseased elderly individuals. Using in vitro three-dimensional (3D) bioengineered skeletal muscle constructs that model aged phenotypes and incorporate a representative extracellular matrix (collagen), are under tension, and display morphological and transcript expression of mature skeletal muscle may more accurately characterize the SkM niche. Furthermore, an in vitro model would provide greater experimental manipulation with regard to gene, pharmacological and exercise (mechanical stretch/electrical stimulation) therapies and thus strategies for combating muscle wasting with age. The present study utilized multiple population-doubled (MPD) murine myoblasts compared with parental controls (CON), previously shown to have an aged phenotype in monolayer cultures (Sharples et al., 2011), seeded into 3D type I collagen matrices under uniaxial tension. 3D bioengineered constructs incorporating MPD cells had reduced myotube size and diameter vs. CON constructs. MPD constructs were characterized by reduced peak force development over 24 h after cell seeding, reduced transcript expression of remodelling matrix metalloproteinases, MMP2 and MMP9, with reduced differentiation/hypertrophic potential shown by reduced IGF-I, IGF-IR, IGF-IEa, MGF mRNA. Increased IGFBP2 and myostatin in MPD vs. CON constructs also suggested impaired differentiation/reduced regenerative potential. Overall, 3D bioengineered skeletal muscle constructs represent an in vitro model of the in vivo cell niche with MPD constructs displaying similar characteristics to ageing/atrophied muscle in vivo, thus potentially providing a future test bed for therapeutic interventions to contest muscle degeneration with age.

Sirtuin 1 regulates skeletal myoblast survival and enhances differentiation in the presence of resveratrol.

Sirtuin 1 also known as NAD-dependent deacetylase sirtuin 1, is a protein that in humans is encoded by the Sirt1 gene. Sirt1 is an enzyme that deacetylates proteins that contribute to cellular regulation and is a key regulator of cell defenses and survival in response to stress. Deletion of Sirt1 abolishes the increase in lifespan induced by calorie restriction or sublethal cytokine stress, indicating that Sirt1 promotes longevity and survival. We have demonstrated that administration of a sublethal dose of tumour necrosis factor-α (TNF-α; 1.25 ng ml(-1)) inhibits myotube formation, and co-incubation with insulin-like growth factor I (IGF-I; 1.5 ng ml(-1)) facilitates C2 myoblast death rather than rescuing differentiation. A higher dose of TNF-α (10 ng ml(-1)) resulted in significant apoptosis, which was rescued by IGF-I (1.5 ng ml(-1); 50% rescue; P < 0.05). We aimed to investigate the role of Sirt1 in the conflicting roles of IGF-I. Quantitative real-time PCR revealed that Sirt1 expression was elevated in myoblasts following incubation of 10 ng ml(-1) TNF-α or 1.25 ng ml(-1) TNF-α plus IGF-I (fivefold and 7.2-fold increases versus control, respectively; P < 0.05). A dose of 10 ng ml(-1) TNF-α induced ∼21 ± 0.7% apoptosis, which was reduced (∼50%; P < 0.05) when administered with IGF-I. Likewise, Sirt1 expression was elevated following 10 ng ml(-1) TNF-α administration, but was reduced (∼30%; P < 0.05) in the presence of IGF-I. C2C12 myoblasts, a subclone of the C2 cell line produced for their differentiation potential and used to examine intrinsic ageing, unlike C2 cells, do not die in the presence of TNF-α and do not upregulate Sirt1. As conditions that induced the greatest myoblast stress/damage resulted in elevated Sirt1 expression, we investigated the effects of Sirt1 gene silencing. Treatment with 10 ng ml(-1) TNF-α or co-incubation with 1.25 ng ml(-1) TNF-α and 1.5 ng ml(-1) IGF-I resulted in apoptosis (20.33 ± 2.08 and 19 ± 2.65%, respectively), which was increased when myoblasts were pretreated with Sirt1 small interfering RNA (31 ± 2.65 and 27.33 ± 2.52%, respectively; P < 0.05) and was reduced (14.33 ± 3.05%, P < 0.05 and 12.78 ± 4.52%, P = 0.054) by resveratrol, which also significantly rescued the block on differentiation. In conclusion, Sirt1 expression increases in conditons of stress, potentially serving to reduce or dampen myoblast death.